Proc. Nati. Acad. Sci. USAVol. 80, pp. 1947-1950, April 1983Cell Biology

Expression of cellular myc and mos genes in undifferentiated B celllymphomas of Burkitt and non-Burkitt types

(onc genes/Burldtt lymphomas/8;14 chromosome translocation/RNA blot analysis)

ROBERT T. MAGUIRE*, TERRY S. ROBINSt, SNORRI S. THORGEIRSSON*, AND CAROLE A. HEILMAN**Laboratory of Carcinogen Metabolism and tLaboratory of Molecular Oncology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Edward V. Evarts, January 10, 1983

ABSTRACT Burkitt lymphomas contain reciprocal translo-cations between chromosome 8 and one of the chromosomes con-taining the immunoglobulin gene loci, prompting speculation thatconsequent activation of a crucial gene(s) on chromosome 8 mightbe involved in the generation of these tumors. Recently the humancounterparts of the retroviral oncogenes v-myc and v-mos havebeen mapped to chromosome 8. We have, therefore, analyzed thelevel of transcription of the cellular myc and mos genes in a varietyof undifferentiated B cell lymphomas of Burkitt and non-Burkitttype that possess either an 8;14 or an 8;22 translocation. These linesexpressed 2- to 5-fold more myc-specific RNA than do B cell lineswithout a translocation. Tumor cell lines of American origin withan 8;14 or 8;22 translocation expressed similar amounts of myc-specific RNA. Tumor cell lines of African origin contained slightlyhigher levels of myc-specific RNA than did those of American or-igin. However, level of expression does not appear to correlatewith the presence or absence of Epstein-Barr virus. Therefore,a major increase in the transcription of this gene secondary totranslocation is unlikely to be the cause of Burkitt lymphoma. Therewas no evidence of mos-related transcripts in any of the cell linestested.

Both Epstein-Barr virus (EBV)-positive and -negative Burkittlymphoma cells carry a specific reciprocal translocation be-tween chromosome 8 and chromosome 14 (1, 2). Recently vari-ant forms of Burkitt lymphoma have been described in whichtranslocations occur between chromosome 8 and chromosomes2 or 22 (3, 4). Studies of somatic cell hybrids between rodentcells and human B cells, in addition to in situ hybridization anal-yses, have shown that the human immunoglobulin heavy chaingene locus is located on human chromosome 14, specifically band14q32 (5, 6). The genes for human A and K immunoglobulin lightchains have been mapped to chromosomes 22 and 2, respec-tively (7, 8). Therefore Burkitt lymphoma translocations involvechromosome 8 and a chromosome carrying an immunoglobulingene. In the Daudi Burkitt lymphoma cell line the break inchromosome 14 has been localized to the variable region of theimmunoglobulin heavy chain (VH region), resulting in the trans-location of a portion of the VH gene region to chromosome 8 (9).Moreover the K immunoglobulin light chain gene is present onthe short arm of chromosome 2 (band p12) in the region wherethe translocation occurs in Burkitt lymphoma variants (10).The consistent involvement ofchromosome 8 in these tumors

has led to the speculation that activation of a cellular gene(s) lo-cated on the distal fragment of chromosome 8 is responsible forthe development of Burkitt lymphoma (11). Recently, the hu-man counterparts of two retroviral oncogenes (c-myc and c-mos)have been mapped to chromosome 8 (12, 13), specifically to re-gions on the long arm of chromosome 8 involved in specific

translocations in Burkitt lymphomas and a variety of other tu-mors (14). Furthermore, it is now known that c-myc is trans-located near the immunoglobulin locus on chromosome 14 inseveral Burkitt lymphoma cell lines (R. Dalla-Favera, personalcommunication). Thus available information is suggestive of in-volvement of c-myc in Burkitt lymphomas.

The Moloney murine sarcoma virus, which contains mos se-quences, produces fibrosarcomas in vivo and transforms fibro-blasts in vitro (15, 16). c-mos-specific RNAs have not been de-tected in uninfected murine cells thus far analyzed (17). How-ever, when the cellular mos sequences of the mouse are linkedto a viral long terminal repeat, they are capable of transformingNIH 3T3 cells in DNA transfection assays (18). The human cel-lular mos gene has been found inactive in DNA transfection ex-periments even when linked to the Moloney murine sarcomavirus long terminal repeat elements (19).

In this reportwe have analyzed the transcriptional activity ofthe human c-myc and c-mos genes in a variety of undifferen-tiated B cell lymphoma lines of Burkitt and non-Burkitt types,in addition to B cell lines that lack the translocations associatedwith these tumors. These include EBV-infected cordblood lym-phocytes and cell lines obtained from patients with infectiousmononucleosis. c-myc-specific RNA was expressed in all linesexamined, with the lymphoma lines consistently expressingslightly higher (2- to 5-fold) levels than lines without any trans-location. There was no evidence of c-mos expression in any ofthe lines tested.

MATERIALS AND METHODSCell Lines. Cell lines used in this study were provided by Ian

Magrath (Pediatric Oncology Branch, National Cancer Insti-tute). Many of the growth and surface marker characteristics ofthese cells have been recently described (20-22). Cells werepassaged in RPMI 1640 medium with glutamine and penicil-lin/streptomycin with 20% fetal calf serum (GIBCO).

Probes and Nick Translation. The 3.5-kilobase (kb) Sac Ifragment of c-myc (chicken) was used in this study. The 2.75-kbEcoRI fragment of hu-mos (human) (pHM2A) was the gift ofGeorge Vande Woude (Laboratory of Molecular Oncology, Na-tional Cancer Institute). The cloning of these genes has beendescribed (19, 23). The probes were nick-translated to specificactivities of approximately 2 x 108 cpm/,ug of cloned DNA asdescribed (24).RNA Isolation, Fractionation, and Hybridization. Total cel-

lularRNA was extracted from approximately 2 x 109 cells in thelogarithmic phase of growth by using a modified guanidine hy-drochloride extraction technique (25). The poly(A)-containingfraction was twice purified by chromatography on oligo(dT)-cel-lulose columns (26).

Abbreviations: EBV, Epstein-Barr virus; EBNA, EBV nuclear antigen;kb, kilobase(s); NaCI/Cit, 0.15 M NaCl/O.015 M sodium citrate.

1947

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

July

23,

202

1

1948 Cell Biology: Maguire et al.

Five micrograms of poly(A)-selected RNAs was fractionatedby electrophoresis on horizontal agarose gels containing 2.2 Mformaldehyde (27, 28) or 10mM methylmercury hydroxide (29).After electrophoresis RNAs were stained in 0.2 M ammoniumacetate containing ethidium bromide (5 ,g/ml), destained afterseveral rinses in distilled water, and photographed under UVlight. RNAs were blotted directly on nitrocellulose filters(Schleicher & Schuell, type HA85) with 20x NaCl/Cit (1XNaCl/Cit is 0.15 M sodium chloride/0.015 M sodium citrate)(30). Filters were baked overnight at 60°C or for 2 hr at 80°C ina vacuum oven. Filter-bound DNAwas hybridized overnight at60°C with the appropriate 32P-labeled probe in a solution con-sisting of 50% (vol/vol) formamide, 5x NaCl/Cit, 0.045 MNa2HPO4, 0.005 M NaH2PO4, denatured yeast RNA (type III)at 400 ug/ml, 0.2% Ficoll, 0.2% polyvinylpyrrolidone, 0.2%bovine serum albumin, and 0.1% sodium dodecyl sulfate.

RESULTSDetection and Quantitation of c-myc RNA in B Cell Lines.

Table 1 summarizes the various B cell lines used in this study.The cell lines are divided into five categories and include EBVnuclear antigen (EBNA)-negative American Burkitt and non-Burkitt lymphoma lines with an 8;14 translocation, EBNA-pos-itive American Burkitt lymphoma lines with an 8;22 translo-cation, EBNA-positive African Burkitt lymphoma lines with an8;14 translocation, EBNA-positive cord blood lymphocyte lines,and EBNA-positive B cell lines from patients in the acute phaseof infectious mononucleosis.To determine the size and relative quantity of c-myc specific

RNAs transcribed in the B cell lines, poly(A)-selected RNA wasisolated from whole cells, denatured, and separated by electro-

Table 1. Derivation and EBNA status of cell lines*Trans-

Cell line Code Classification EBNA locationLandis JLPC 119 American Burkitt - 8;14

lymphomaJD-PB JD 38 Undifferentiated - 8;14

lymphomaJD-A JD 39 Undifferentiated - 8;14

lymphomaBM-1 PA 682 BM 1 American Burkitt + 8;22

lymphomaBM-2 PA 682 BM 2 American Burkitt + 8;22

lymphomaPE-1 PA 682 PE 1 American Burkitt + 8;22

lymphomaPE-2 PA 682 PE 2 American Burkitt + 8;22

lymphomaRaji Raji African Burkitt + 8;14

lymphomaDaudi Daudi African Burkitt + 8;14

lymphomaNamalva Namalva African Burkitt + 8;14

lymphomaEckhert IME 182 Infectious mono- + -

nucleosisHotz IMH 182 Infectious mono- + -

nucleosisBell IMB 182 Infectious mono- + -

nucleosisCB 23 CB 23 Cord blood lym- + -

phocytes* I. T. Magrath and J. Whang-Peng, personal communication; and seerefs. 20-22.

phoresis through formaldehyde/agarose gels. The gels werestained with ethidium bromide to verify that equal concentra-tions of intact RNA were used in comparative analyses prior totransfer onto nitrocellulose membranes as described in Mate-rials and Methods. The transfer of RNA species onto nitrocel-lulose was complete for species less than 4.8 kb.

The c-myc probe detected a major 2.7-kb transcript in all linestested (Fig. 1). Occasionally minor bands of various sizes be-tween 5 and 6 kb were detected with this probe, but they werenot considered further due to their highly inconsistent pres-ence. The intensity of the 2.7-kb band was slightly increased (2-to 5-fold) in the tumor lines when compared to the transloca-tion-negative cord blood and infectious mononucleosis lines.These results were confirmed in experiments in which a 2- to5-fold dilution of tumor cell RNA was necessary to bring the in-tensity of the c-myc band to the level of control RNA (Fig. 2).The lymphomas of American origin expressed similar amountsof c-myc-specific RNA whether they possessed an 8;14 or an8;22 translocation (compare the first three lanes with the last twolanes in Fig. 1). The tumor lines of African origin expressedslightly more c-myc-related RNA than those ofAmerican origin.No comparable amplification of the human c-myc-specificDNAsequences was demonstrated (data not shown). The presence ofEBV does not appear to directly enhance c-myc expression asdemonstrated by a comparison of EBNA-positive and -negativeAmerican lymphoma lines. However, the absence of EBNAexpression in cell lines does not necessarily preclude the pres-ence of EBV DNA sequences other than those encoding theEBNA gene product. c-myc expression in the HL60human pro-myelocytic leukemiacell line is increased approximately 10-foldover control cell lines, as previously reported (31).

Lack of c-mos RNA Expression in Human B Cell Lines. Todetermine the relative expression of c-mos RNA in the B cell

U1) -maC,,z

:J n

c-ow

Y

-)I W

N N- 0

o m c: <I 0 0 a

k b

m.

FIG. 1. Hybridization of c-myc (chicken) to poly(A)-selected RNAs.Poly(A)-selected RNAs (5 ug) were fractionated by electrophoresis onhorizontal agarose gels containing 2.2 M formaldehyde. RNAs weretransferred to a nitrocellulose filter and hybridized to a32P-labeled clonednick-translated c-myc probe. Landis, JD-PB, andJD-A are EBNA-neg-ative tumor cell lines of American origin containing an 8;14 translo-cation. Eckhert and Hotz are EBNA-positive translocation-negative Bcell lines from patients with infectious mononucleosis. CB 23 isan EBNA-positive translocation-negative cord blood B lymphocyte line. Raji andDaudi are EBNA-positive African Burkitt lymphoma lines that con-tain an 8;14 translocation. BM-1 and BM-2 are separate lines derivedfrom the same patient and are EBNA-positive American Burkitt lym-phoma lines with an 8;22 translocation. HL-60 is a human promyelo-cytic leukemia cell line and was the gift of R. C. Gallo (National CancerInstitute).

Proc. Natl. Acad. Sci. USA 80 (1983)

i I.'...

2.7- '40."

Dow

nloa

ded

by g

uest

on

July

23,

202

1

Proc. Natl Acad. Sci. USA 80 (1983)

DA U DI

RNA (gg) 5 2.5 1

28S -

1 8S-

-...

28S-

1a8S-

RAJ I2.5

I

H1 0 T Z5 2.5

hh1r.-ii

FIG. 2. Hybridization of c-myc (chicken) to dilutions of poly(A)-selected RNAs. Various amounts (5, 2.5, or 1 ug) of poly(A)-selected RNAs werefractionated by electrophoresis on horizontal agarose gels containing 2.2 M formaldehyde. RNAs were stained with ethidium bromide and pho-tographed prior to transfer to nitrocellulose filters and hybridization to nick-translated c-myc probe. Ethidiumbromide stainedRNAs pictured abovecorrespond to RNAs directly below, which were hybridized to the c-myc probe.

lines, poly(A)-selected RNA isolated from whole cellsDNA were denatured and separated by electrophoimethylmercury/agarose gels and treated as descrNo c-mos-specific RNA was detected in any of th

(Fig. 3), although the c-mos (human) probe was c-tecting as little as 25 pg of c-mos-containing DNA. Iof intact RNA species was also verified by rehylthese filters with c-myc, again revealing the presE

pHM2A DNA

)CD

CDncC CLC LO CqUL N 0

N o 6

4c

-j

i

m iX CL z

0

03

FIG. 3. Lack of hybridization of c-mos (human) to pRNAs. Poly(A)-selected RNAs (5 fig) and 2.5, 0.25, a]pHM2A DNA were fractionated by electrophoresis on hiose gels containing 10 mM methylmercury hydroxidtransferred to a nitrocellulose filter and hybridized to 1nick-translated pHM2A c-mos-containing probe. BM-1,1 are separate lines derived from the same patient. Cescribed in Table 1.

sandpHM2Aresis throughibed above.ie lines testedapable of de-

myc-specific 2.7-kb transcript (data not shown). The hu-mos 2.75-kb EcoRI DNA fragment was identified in all cell lines (data notshown).

DISCUSSION[he presence The recent localization of the human c-myc and c-nos genes to)ridization of chromosome 8 has led to speculation that activation of one ormnce of the c- both of these genes by translocation might be involved in the

genesis of Burkitt lymphomas. Activation of c-myc by avian leu-kosis virus is associated with B cell lymphomas of the bursa inchickens (32), a tumor similar in many respects to Burkitt lym-

w phomas ofman. Recently Dalla-Faveraetal. have demonstratedthat c-myc (human) is translocated very near the immunoglob-

4 UJ ulinheavy chain locus on chromosome 14 in several Burkitt lym-rrmu phoma lines studied thus far (R. Dalla-Favera, personal

munication). Transfection studies with NIH 3T3 mouse cells,however, do not yield c-myc as the transforming element fromchicken bursal lymphomas (33). We have similarly failed to findevidence of human c-myc sequences in DNAs from NIH 3T3primary transfectants in which the transformed phenotype hasbeen induced by Burkitt lymphoma DNAs, though the murinec-myc sequences can be easily detected (unpublished obser-vation). These data indicate that although activation of c-myc

may be involved in the oncogenesis of certain B cell lympho-mas, other cellular gene(s) derived from either bursal or Burkittlymphomas have potential oncogenic activity. c-myc activationtherefore may represent a necessary event in a multistage pro-

cess leading to tumor formation.Our data demonstrate a 2- to 5-fold increase in c-myc expres-

sion by Burkitt lymphoma lines when compared to EBV-"im-mortalized" B cell lines. No comparable amplification of the hu-man myc-specific DNA sequence was demonstrated (data not

oly(A)-selected shown). Tumor cell lines of American origin with 8;14 or 8;22nd 0.025 ng of translocations express similar amounts ofc-myc RNA. The pres-

Lorizontal agar- ence or absence of EBV does not correlate with the level of c-

bhe 32P-labeled myc expression. The slight increase in c-myc RNA expression inPE-2, and PE- African lines when compared to those of American origin could11 lines are de- possibly be explained by the slightly different stage of B cell

differentiation that these tumors represent (34).

-2.7 kb

Cell Biology: Maguire et al. 1949

5

4

Dow

nloa

ded

by g

uest

on

July

23,

202

1

1950 Cell Biology: Maguire et aL

In avian leukosis virus-induced bursal lymphomas of thechicken, c-myc RNA expression is at least 30-fold increasedover control tissues (32). In the HL-60 promyelocytic leukemiacell line c-myc RNA expression is increased 10-fold over controls(31), apparently due to an amplification of the c-myc gene (35,36). This amplification is also present in the primary leukemiacells of the patient from which the line was derived, suggestingthat increased levels of c-myc expression may have been in-volved in the leukemic transformation of this patient's cells (36).The small and variable increase in c-myc expression among

the lymphomas examined in the present study argues againstincreased transcription of this gene being the cause of Burkitttumors. In addition it seems that c-myc may be translocated tothe inactive unrearranged chromosome 14 in these tumors (9),making "up-regulation" by insertion near an active promoterregion less likely. The lack of control populations of B cells atthe same stage of differentiation as Burkitt tumor cells furtherchallenges the significance of the small differences in c-mycexpression seen here. Changes in c-myc gene environment sec-ondary to translocation might lead to a new or altered promoterregion no longer subject to normal regulatory controls. The al-tered regulation of this gene without markedly increasing itstranscription might be associated with tumor formation. Subtlequalitative differences in the c-myc transcript in Burkitt tumorsis another possibility that cannot be ruled out by our experi-ments. Alternatively, c-myc may not be involved in the gen-eration of Burkitt lymphomas, its translocation in these tumorsbeing a coincidental finding.

There is no evidence of c-mos transcription in our cell lines,and it is therefore unlikely that this gene plays a role in the gen-eration of Burkitt lymphomas.We thank Eric Westin for his critical review of this manuscript and

Larry Linder for his excellent technical assistance. We thank FrancesWilliams for assistance in preparation of the manuscript.

1. Manolov, G. & Manolova, Y. (1972) Nature (London) 237, 33-34.2. Zech, L., Haglund, U., Nilsson, K. & Klein, G. (1976) Int.J. Can-

cer 17, 47-56.3. Van denBerghe, H., Parloir, C., Gosseye, S., Englebienne, U.,

Cornu, G. & Sokal, G. (1979) Cancer Genet. Cytogenet. 1, 9-14.4. Bernheim, A., Berger, R. & Lenoir, G. (1981) Cancer Genet. Cy-

togenet. 3, 307-316.5. Croce, C. M., Shander, M., Martinis, J., Circurel, L., D'Ancon-

na, G. G., Dolby, T. W. & Koprowski, H. (1979) Proc. Natl Acad.Sci. USA 76, 3416-3419.

6. Kirsch, I. R., Morton, C. C., Nakahara, K. & Leder, P. (1982)Science 216, 301-303.

7. Erikson, J., Martinis, J. & Croce, C. M. (1981) Nature (London)294, 173-175.

8. McBride, 0. W., Hieter, P. A., Hollis, G. F., Swan, D., Otey,M. C. & Leder, P. (1982)J. Exp. Med. 155, 1480-1490.

9. Erikson, J., Finan, J., Nowell, P. C. & Croce, C. M. (1982) Proc.NatL Acad. Sci. USA 79, 5611-5615.

10. Malcolm, S., Barton, P., Murphy, C., Ferguson-Smith, M. A.,Bentley, D. L. & Rabbitts, T. H. (1982) Proc. Natl Acad. Sci. USA79, 4957-4961.

11. Klein, G. (1981) Nature (London) 294, 313-318.12. Dalla-Favera, R., Bregni, M., Erikson, J., Patterson, D., Gallo,

R. C. & Croce, C. M. (1982) Proc. Natl Acad. Sci. USA 79, 7824-7827.

13. Prakash, K., McBride, 0. W., Swan, D. C., Devare, S. G., Tro-nick, S. R. & Aaronson, S. A. (1982) Proc. Natl Acad. Sci. USA 79,5210-5214.

14. Neel, B. G., Jhanwar, S. C., Chaganti, R. S. K. & Hayward, W.S. (1982) Proc. Natl Acad. Sci. USA 79, 7842-7846.

15. Moloney, J. B. (1966) Natl Cancer Inst. Monogr. 22, 139-142.16. Aaronson, S. A., Jainchill, J. L. & Todaro, G. (1970) Proc. Natl.

Acad. Sci. USA 66, 1236-1243.17. Gattoni, S., Kirschmeier, P., Weinstein, J., Escobedo, J. & Dina,

D. (1982) Mol CelL Biol. 2, 42-51.18. Blair, D. G., Oskarsson, M., Wood, T. G., McClements, W. L.,

Fischinger, P. J. & Vande Woude, G. F. (1981) Science 212, 941-943.

19. Watson, R., Oskarsson, M. & Vande Woude, G. F. (1982) Proc.Natl Acad. Sci. USA 79, 4078-4082.

20. Benjamin, D., Magrath, I. T., Maguire, R., Janus, C., Todd, H.D. & Parsons, R. G. (1982)J. Immunol. 129, 1336-1342.

21. Magrath, I. T., Pizzo, P. A., Whang-Peng, J., Douglas, E. C., Al-abaster, O., Gerber, P., Freeman, C. B. & Novikovs, L. (1980)J.Nati Cancer Inst. 64, 465-476.

22. Magrath, I. T., Freeman, C. B., Pizzo, P. A., Gadek, J., Jaffe,E., Santaella, M., Hammer, C., Frank, M., Reaman, G. & No-vikovs, L. (1980) J. Natl, Cancer Inst. 64, 477-483.

23. Robins, T., Bister, K., Garon, C., Papas, T. & Duesberg, P. (1982)J. Virol 41, 635-642.

24. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977)J.Mol Biol 113, 237-251.

25. Deeley, R. G., Gordon, J. I., Burns, A. T. H., Mullinix, K. P.,Bina-Stein, M. & Goldberger, R. F. (1977) J. Biol. Chem. 252,8310-8319.

26. Aviv, H. & Leder, P. (1972) Proc. Natl Acad. Sci. USA 69, 1408-1412.

27. Rave, N., Crkvenjakov, R. & Boedtker, H. (1979) Nucleic AcidsRes. 6, 3559-3567.

28. Heilman, C. A., Engel, L., Lowy, D. R. & Howley, P. M. (1982)Virology 119, 22-34.

29. Bailey, J. M. & Davidson, N. (1976) Anal Biochem. 70, 75-85.30. Thomas, P. S. (1980) Proc. NatW Acad. Sci. USA 77, 5201-5205.31. Westin, E. W., Wong-Staal, F., Gelmann, E. P., Dalla-Favera,

R., Papas, T. S., Lautenberger, J. A., Eva, A., Reddy, E. P.,Tronick, S. R., Aaronson, S. A. & Gallo, R. C. (1982) Proc. NatWAcad. Sci. USA 79, 2490-2494.

32. Hayward, W. S., Neel, B. G. & Astrin, S. A. (1981) Nature (Lon-don) 290, 475-480.

33. Cooper, G. M. & Neiman, P. E. (1981) Nature (London) 292, 857-858.

34. Freeman, C. B., Magrath, I. T., Benjamin, D., Makuch, R.,Douglas, E. C. & Santaella, M. L. (1982) Clin. Immunol Immu-nopath. 25, 103-111.

35. Collins, S. & Groudine, M. (1982) Nature (London) 298, 679-681.36. Dalla-Favera, R., Wong-Staal, F. & Gallo, R. C. (1982) Nature

(London) 299, 61-63.

Proc. Nad Acad. Sci. USA 80 (1983)

Dow

nloa

ded

by g

uest

on

July

23,

202

1